Isolation and partial characterization of proteins from Tinospora cordifolia against Pseudomonas aeruginosa PAO1

Tinospora cordifolia is a deciduous shrub. It is a well recognized for its medicinal properties in Indian Ayurveda system. It is one of the most versatile shrub commonly known as “giloy”. The plant gained attention due to its various biological activities such as anti-diabetic, anti-microbial, anti-allergic, anti-oxidant and anti-cancer activities. The plant parts contain various chemical constituents such as alkaloids, steroids, glycosides and polysaccharides. The presence of high levels of polyphenols, mucilage, and other components often leads to protein precipitation hence reduced solubility. Therefore, the present study was undertaken for isolation and purification of the antibacterial protein of T. cordifolia collected from Hamirpur district (Neri), Himachal Pradesh. Analysis was done by methanolic, methanolic, extract of samples taken from Neri village. It was found that methanol act as better solvent for extraction of proteins. Ammonium sulphate precipitation of methanolic crude extract showed that 60-70% precipitation was found best for precipitation of proteins. Further, 60-70% precipitates were subjected for dialysis followed by ion exchange chromatography. The fractions obtained after ion exchange chromatograph were assayed by antibacterial activity against test organism. The active fractions were pooled together and carried out for SDS-PAGE. The total protein content present in methanol in the crude extract was 2.056mg/ml. The ammonium sulphate precipitation showed 10.833±0.928mm zone of inhibition and after purification the antibacterial activity increased to 15±0.289 mm. The partial characterization of proteins revealed that the antibacterial proteins was stable at optimal pH 8, heat stable at 40 ℃ temperature and stable at low salt concentration. For future aspects, the stem extract and protein can be further exploited for its other properties such as it sequencing.